Force scanning probe microscope

ABSTRACT

A force scanning probe microscope (FSPM) and associated method of making force measurements on a sample includes a piezoelectric scanner having a surface that supports the sample so as to move the sample in three orthogonal directions. The FSPM also includes a displacement sensor that measures movement of the sample in a direction orthogonal to the surface and generates a corresponding position signal so as to provide closed loop position feedback. In addition, a probe is fixed relative to the piezoelectric scanner, while a deflection detection apparatus is employed to sense a deflection of the probe. The FSPM also includes a controller that generates a scanner drive signal based on the position signal, and is adapted to operate according to a user-defined input that can change a force curve measurement parameter during data acquisition.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of Ser. No. 10/006,085, filed Dec. 6,2001, entitled “Force Scanning Probe Microscope”, which is expresslyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to probe microscopes, and moreparticularly, a probe microscope (PM) apparatus and method for sensingtip-sample interaction forces.

2. Description of Related Art

Developments in nanotechnology have enabled mechanical experiments on abroad range of samples including single molecules, such that fundamentalmolecular interactions can be studied directly. The mechanicalproperties of biological molecules, in particular, such as actinfilaments and DNA has lead to the development of a range ofinstrumentation for conducting these studies. In this regard, systemsand methods differing in force and dynamic ranges currently being usedinclude magnetic beads, optical tweezers, glass microneedles,biomembrane force probes (BFP), scanning probe microscopy (SPM), andatomic force microscopy (AFM).

With a force sensitivity on the order of a few pico-Newtons (pN=10⁻¹²N),an AFM is an excellent tool for probing fundamental force interactionsbetween surfaces. AFM has been used to probe the nature of attractivevan der Waals and attractive/repulsive electrostatic forces betweensystems such as metal probes and insulating mica surfaces, andinsulating probes on insulating and conducting samples with materialssuch as silicon nitride, diamond, alumina, mica, glass and graphite.Other applications include the study of adhesion, friction, and wear,including the formation or suppression of capillary condensation onhydrophilic silicon, amorphous carbon and lubricated SiO₂ surfaces.

More particularly, for biological molecules, force is often an importantfunctional and structural parameter. Biological processes such as DNAreplication, protein synthesis, drug interaction, to name a few, arelargely governed by intermolecular forces. However, these forces areextremely small. With its sensitivity in the pico-Newton scale, the SPMhas been employed to analyze these interactions. In this regard, SPMstypically are used to generate force curves that provide particularlyuseful information for analyzing very small samples.

The knowledge regarding the relation between structure, function andforce is evolving and therefore single molecule force spectroscopy,particularly using SPM, has become a versatile analytical tool forstructural and functional investigation of single bio-molecules in theirnative environments. For example, force spectroscopy by SPM has beenused to measure the binding forces of different receptor-ligand systems,observe reversible unfolding of protein domains, and investigatepolysaccharide elasticity at the level of inter-atomic bond flips.Moreover, molecular motors and their function, DNA mechanics and theoperation of DNA-binding agents such as proteins in drugs have also beenobserved. Further, the SPM is capable of making nano-mechanicalmeasurements (such as elasticity) on biological specimens, thusproviding data relative to subjects such as cellular and proteindynamics.

Another main application of making AFM force measurements is inmaterials science where the study of mechanical properties of nano-scalethin films and clusters is of interest. For example, as microstructuressuch as integrated circuits continue to shrink, exploring the mechanicalbehavior of thin films from known properties of the materials becomesincreasingly inaccurate. Therefore, continuing demand for fastercomputers and larger capacity memory and storage devices placesincreasing importance on understanding nano-scale mechanics of metalsand other commonly used materials.

PMs, including instruments such as the atomic force microscope (AFM),are devices that typically use a sharp tip and low forces tocharacterize the surface of a sample down to atomic dimensions.Generally, AFMs include a probe having a tip that is introduced to asurface of a sample to detect changes in the characteristics of thesample. In this case, relative scanning movement between the tip and thesample is provided so that surface characteristic data can be acquiredover a particular region of the sample, and a corresponding map of thesample surface can be generated. However, PMs also include devices suchas molecular force probes (MFPs) that similarly use a probe tocharacterize sample properties but do not scan.

In one application of AFM, either the sample or the probe is translatedup and down relatively perpendicularly to the surface of the sample inresponse to a signal related to the motion of the cantilever of theprobe as it is scanned across the surface to maintain a particularimaging parameter (for example, to maintain a set-point oscillationamplitude). In this way, the feedback data associated with this verticalmotion can be stored and then used to construct an image of the samplesurface corresponding to the sample characteristic being measured, e.g.,surface topography. Other types of images are generated directly fromthe detection of the cantilever motion or a modified version of thatsignal (i.e., deflection, amplitude, phase, friction, etc.), and arethus not strictly topographical images.

In addition to surface characteristic imaging such as topographicalimaging, the AFM can probe nano-mechanical and other fundamentalproperties of samples and their surfaces. Again, AFM applications extendinto applications ranging from measuring colloidal forces to monitoringenzymatic activity in individual proteins to analyzing DNA mechanics.

When measuring biological samples, it is useful to measure, for example,the stiffness of the sample; in one example, to separate salt crystalsfrom DNA or to separate the DNA from a hard surface. In U.S. Pat. No.5,224,376, assigned to the assignee of the present invention, an atomicforce microscope is described in which the system can map both the localthe stiffness (force spectroscopy) and the topography of a sample. Inthe preferred implementation, a stiffness map of the sample is obtainedby modulating the force between the tip and sample during a scan bymodulating the vertical position of the sample while keeping the averageforce between the tip and the sample constant. The bending of thecantilever, which is a measure of the force on the tip, is measured byan optical detector that senses the deflection of a light beam reflectedfrom the back of the cantilever. In a simple example, the AFM and forcespectroscopy apparatus of this patent has been used to study DNA layingon a glass surface. Modulating the force and then imaging the stiffnessof the sample has the advantage that a surface such as glass, which hasa rough topographic image, will have a flat stiffness image, permittingsoft molecules on it such as DNA to be readily imaged.

Notably, a key element of the probe microscope is its microscopicsensor, i.e., the probe. The probe includes a microcantilever, thedesign and fabrication of which is well-known in the field, which istypically formed out of silicon, silicon nitride, or glass, and hastypical dimensions in the range of 10-1000 microns in length and 0.1-10microns in thickness. The probe may also include a “tip,” which,particularly in AFM, is typically a sharp projection near the free endof the cantilever extending toward the sample. In the more general fieldof probe microscopy, the tip may be absent or of some other shape andsize in order to control the particular type, magnitude, or geometry ofthe tip-sample interaction or to provide greater access to chemicallymodify the tip surface.

The second key element of a probe microscope is a scanning mechanism(“the scanner”), which produces relative motion between the probe andthe sample. It is well-known by those in the field that such scannersmay move either the tip relative to the sample, the sample relative tothe tip, or some combination of both. Moreover, probe microscopesinclude both scanning probe microscopes in which the scanner typicallyproduces motion in three substantially orthogonal directions, andinstruments with scanners that produce motion in fewer than threesubstantially orthogonal directions (i.e.—MFP).

Turning to FIGS. 1A-1E and 2, force spectroscopy using SPM isillustrated. More particularly, FIGS. 1A-1E show how the forces betweena tip 14 of a probe 10 and a sample 16, at a selected point (X,Y) on thesample, deflect a cantilever 12 of probe 10 as the tip-sample separationis modulated in a direction generally orthogonal to the sample surface.FIG. 2 shows the magnitude of the forces as a function of sampleposition, i.e., a force curve or profile.

In FIG. 1A, probe 10 and sample 16 are not touching as the separationbetween the two is narrowed by moving the sample generally orthogonallytoward the sample surface. Zero force is measured at this point of thetip-sample approach, reflected by the flat portion “A” of the curve inFIG. 2. Next, probe 10 may experience a long range attractive (orrepulsive force) and it will deflect downwardly (or upwardly) beforemaking contact with the surface. This effect is shown in FIG. 1B. Moreparticularly, as the tip-sample separation is narrowed, tip 14 may“jump” into contact with the sample 16 if it encounters sufficientattractive force from the sample. In that case, the correspondingbending of cantilever 12 appears on the force profile, as shown in FIG.2 at the curve portion marked “B.”

Turning next to FIG. 1C, once tip 14 is in contact with sample 16, thecantilever will return to its zero (undeflected) position and moveupwardly as the sample is translated further towards probe 10. Ifcantilever 12 of probe 10 is sufficiently stiff, the probe tip 14 mayindent into the surface of the sample. Notably, in this case, the slopeor shape of the “contact portion” of the force curve can provideinformation about the elasticity of the sample surface. Portion “C” ofthe curve of FIG. 2 illustrates this contact portion.

In FIG. 1D, after loading cantilever 12 of probe 10 to a desired forcevalue, the displacement of the sample 16 is reversed. As probe 10 iswithdrawn from sample 16, tip 14 may either directly adhere to thesurface 16 or a linkage may be made between tip 14 and sample 16, suchas via a molecule where opposite ends are attached to the tip 14 andsurface 16. This adhesion or linkage results in cantilever 14 deflectingdownwards in response to the force. The force curve in FIG. 2illustrates this downward bending of cantilever 14 at portion “D.”Finally, at the portion marked “E” in FIG. 2, the adhesion or linkage isbroken and probe 10 releases from sample 16, as shown in FIG. 1E.Particularly useful information is contained in this portion of theforce curve measurement, which contains a measure of the force requiredto break the bond or stretch the linked molecule.

An example of a sample force measurement as described above is shown inFIG. 3 where two complimentary strands of DNA 20 are immobilized on thetip and sample surfaces 22 and 24, respectively. By modulating thetip-sample separation, a force curve such as that shown in FIG. 2 can begenerated. As a result, a quantitative measurement of the forces andenergetics required to stretch and un-bind the DNA duplexes can bemapped.

In sum, a simple force curve records the force on the tip of the probeas the tip approaches and retracts from a point on the sample surface. Amore complex measurement known as a “force volume,” is defined by anarray of force curves obtained as described above over an entire samplearea. Each force curve is measured at a unique X-Y position on thesample surface, and the curves associated with the array of X-Y pointsare combined into a 3-dimensional array, or volume, of force data. Theforce value at a point in the volume is the deflection of the probe atthat position (x, y, z).

Although this example relates specifically to AFM force measurementsthat use cantilever deflection as a measure of force, those skilled inthe art will recognize that there are other physico-chemical propertiesthat can be measured using substantially similar probes,instrumentation, and algorithms.

Although SPMs are particularly useful in making such measurements, thereare inherent problems with known systems. In particular, typical SPMsuse conventional fine motion piezoelectric scanners that translate thetip or sample while generating topographic images and making forcemeasurements. A piezoelectric scanner is a device that moves by amicroscopic amount when a voltage is applied across electrodes placed onthe piezoelectric material of the scanner. Overall, the motion generatedby such piezoelectric scanners is not entirely predictable, and hencesuch scanners have significant limitations.

A conventional AFM 30 including a piezoelectric scanner 32 is shown inFIG. 4. Scanner 32 is a piezoelectric tube scanner including an X-Ysection 34 and a Z section 36. In this arrangement, Z section 36 ofscanner 32 is adapted to support a sample 42.

To make a force measurement, section 34 of scanner 32 translates sample42 relative to probe 44 of AFM 30 to a selected position (x,y). As notedpreviously, to actuate scanner 32, sections 34, 36 include electrodesplaced thereon (such as 38 and 40 for the X-Y section) that receiveappropriate voltage differentials from a controller that, when applied,produce the desired motion. Next, Z section 36 is actuated to translatesample 42 toward a tip 46 of probe 44, as described in connection withthe force curve measurement shown in FIGS. 1A-1E and 2. Again, as tip 46interacts with sample 42, a cantilever 48 of probe 44 deflects. Thisdeflection is measured with a deflection detection system 50. Detectionsystem 50 includes a laser 51 that directs a light beam “L” towards theback of cantilever 48, which is reflective. The beam “L” reflects fromcantilever 48, and the reflected beam “L” contacts a beam steeringmirror 52 which directs the beam “L” towards a sensor 54. Sensor 54, inturn, generates a signal indicative of the cantilever deflection.Because cantilever deflection is related to force, the deflectionsignals can be converted and plotted as a force curve.

Standard piezoelectric scanners for SPMs usually can translate in threesubstantially orthogonal directions, and their size can be modified toallow scan ranges of typically several nanometers to several hundredmicrons in the X-Y plane and typically <10 microns in the Z-axis.Moreover, depending on the particular implementation of the AFM, thescanner is used to either translate the sample under the cantilever orthe cantilever over the sample.

The methods and limitations described above pertaining to currenttypical scanners in SPM are in many cases acceptable in applicationswhere a probe microscope is being used in conventional imaging modes inwhich the XY motion is typically periodic and it is acceptable to use arelative measure of Z movement.

However force spectroscopy experiments typically demand more precisecontrol of relative tip-sample motion, particularly in the Z axis (theaxis substantially perpendicular to the sample surface).

Typical piezoelectric scanners do not exhibit linear motion, i.e., agiven change in the applied drive voltage to the piezo will result in adifferent magnitude of motion in different areas if the operating range.Typical piezoelectric scanners also commonly exhibit hysteretic motion,i.e., if a particular voltage ramp is applied to the scanner and thenthe ramp is re-traced exactly in reverse, one finds that the scannerfollows a different position path on the extend versus the retract.Piezoelectric scanners also “creep,” which means that they continue toextend or retract for a period of time after the applied drive voltagehas stopped changing. Piezoelectric tube scanners also typically havelow resonant frequencies in the Z-axis. Those skilled in the artrecognize that this represents a serious limitation on the range ofoperating speeds for which the scanner is useful. This is because thepiezoelectric material undergoes complex oscillatory motion when passingthrough and near the resonant frequency.

Any one or more of these limitations clearly jeopardize the integrity ofthe tip-sample motion, and therefore the corresponding data collected isof marginal usefulness. Overcoming these limitations is one of the keygoals of this invention.

Alternative means of relative tip-sample motion exist that address theseconcerns, although they can create new problems. For instance, sensorscan be coupled to piezoelectric scanners by various means well-known inthe field. Such sensors can produce a more accurate record of motioncompared to the more usual assumption that the control voltage isrepresentative of the motion. However, adding sensors to a scanner onlydetects, not corrects, these undesirable motions. However, such sensoredscanners can be used in a closed-loop feedback configuration in whichthe motion is monitored during a change in position and the applieddrive voltage is modified as necessary to make the actual path of motionmore closely match the path specified by the control input signal. Suchsensored and closed-loop scanners are most commonly implemented inconjunction with a different mechanical design of the scanner known as apiezo-actuated flexure stage (“stage”). These stages contain mechanicalconstraints (flexures) on the motion of the stage intended primarily toconstrain the motion of the stage to one axis and to mechanicallystiffen the stage. This design also presents more obvious possibilitiesfor incorporating a sensor than piezoelectric tube designs, althougheither is feasible in practice. The flexure stage offers the additionaladvantage of increasing the resonant frequency of the stage relative toa piezoelectric tube scanner with similar range.

Nevertheless, although the above may seem to suggest a design includingclosed-loop flexure stages in all three axes, in practice such a designhas significant drawbacks. Among the disadvantages of a three-flexurestage design, is that 3-axis flexure stages are much larger than atypical piezoelectric tube scanner of similar range due to the addedmass and volume of the constraining mechanism and sensors. In practice,larger designs more readily couple outside vibrational and acousticnoise sources into the motion of the scanner, which significantlydegrades the scanners usefulness for force spectroscopy. Closed-loopflexure stages are also significantly more expensive than piezoelectrictube scanners of similar range.

Therefore, the use of flexure stages for all three axes is not desirablefor the design of a compact, low-noise, relatively inexpensiveinstrument.

There are also drawbacks associated with the methods employed to makeconventional force curve measurements. Experimentally, a force curvemeasurement is made by applying, for example, a cyclical triangle wavevoltage pattern to the electrodes of the Z-axis scanner as shown in FIG.5A. The triangle wave drive signal causes the scanner to expand and thencontract in the vertical direction, generating relative motion betweenthe probe and the sample. In such a system, the amplitude of thetriangle wave as well as the frequency of the wave can be controlled sothat the researcher can linearly vary the distance and speed that theAFM cantilever tip travels during the force measurement. In FIG. 5B, adrive signal similar to that shown in FIG. 5A is illustrated. However,in this case, the drive signal includes a pause between each change inthe direction of Z scanner motion. In each case, the drive signal iscyclical. However, oftentimes it is desired to modify the parameters ofthe force measurement in a non-cyclical manner, including the speed atwhich the tip-sample separation is modulated, the duration of a pause(to allow molecular binding between tip and molecules on the surface,for example), etc. to analyze forces corresponding to, for example,complex mechanical models of certain samples. In this regard it isnotable that conventional systems often lack flexibility in makingmeasurements that are non-cyclic. Therefore, a system was desired inwhich the flexibility in performing the force measurement is improved.For example, a specific change or rate of change in tip-sample force ora specific value of a tip-sample force may indicate some propertypertaining to the sample in question. In response, it would be desirableto alter a force curve measurement parameter (such as the speed of themovement) in response to a specific measurement condition. Or, forexample it may be desirable to instead of following a path of position(separation) versus time, follow a path of force versus time where theposition (separation) is controlled to produce the desired forceprofile.

Overall, the field of making force measurements with a probe microscopewas in need of a system including a scanner that is contained in arelatively small package, provides a large Z-axis range with accuratecontrol of Z motion, and has a relatively high resonance frequency.Moreover, the field was in need of a system capable of performing forcemeasurements according to particular forces measured, and according toparticular profiles to maximize the flexibility of making the forcemeasurement. Although not a fundamental requirement for forcespectroscopy, SPM-based system may also be made capable of makingconventional AFM measurements (e.g., topography) by simply switchingmodes of operation.

SUMMARY OF THE INVENTION

The preferred embodiment overcomes the drawbacks of prior art systems byproviding a force scanning probe microscope (FSPM) that combines aflexured Z stage and a piezoelectric tube XY scanner. More particularly,the Z actuator stage is sensored so that proper tip-sample positioningis maintained, thus maximizing the integrity of the force curvemeasurements. Moreover, the FSPM takes advantage of the ability to makeimproved force measurements with more exact positioning by also beingadapted to operate according to user-defined position/force profiles, aswell as having the ability to change a force measurement parameter inresponse to a trigger condition. In addition to these automatic controlfeatures, the FSPM also includes a manual control device that allows auser to manipulate tip-sample separation according to an alert feedback,such as a tactile or audio alert. Although not a fundamental requirementfor force spectroscopy, the probe microscope based FSPM may also be madecapable of operating in conventional AFM modes.

According to a first aspect of the preferred embodiment, a piezoelectricscanner includes a piezoelectric tube that generates scanner motion intwo substantially orthogonal axes defining a substantially planarsurface (“the scan plane”). In addition, the scanner includes a flexuredpiezoelectric stage that generates scanner motion in a third axissubstantially orthogonal to the scan plane. Moreover, the piezoelectricstage is preferably coupled to the piezoelectric tube.

According to another aspect of the preferred embodiment, the sensorincludes a displacement sensor that detects motion in the third axis andgenerates a corresponding position signal.

In another aspect of this embodiment, the scanner is disposed in a probemicroscope having a probe and a detection apparatus that senses motionof the probe. A sample is positioned such that the scanner producesrelative tip-sample motion, and the displacement sensor is mounted onthe stage.

According to a still further aspect of the preferred embodiment, thestage and sensor are connected to a data acquisition and control system,and wherein the data acquisition and control system generates a controlsignal that drives the stage. Also, the control signal is preferablygenerated in response to a user input, where the user input can be aposition or force profile, which may include triggers.

According to an alternate aspect of the preferred embodiment, the userinput corresponds to a desired scanner motion, and the control signaldrives the stage so the scanner motion in the third axis is generallythe same as the desired scanner motion.

According to a still further aspect of the preferred embodiment, a forcescanning probe microscope (FSPM) includes a piezoelectric scanner havinga surface that supports the sample so as to move the sample in threesubstantially orthogonal directions. The FSPM also includes adisplacement sensor that measures movement of the sample in a directionsubstantially orthogonal to the surface and generates a correspondingposition signal so as to provide closed loop position feedback. Inaddition, a probe is fixed relative to the piezoelectric scanner, whilea probe motion detection apparatus is employed to sense motion of theprobe. The FSPM also includes a data acquisition and control system thatgenerates a scanner drive signal based on the position signal and auser-defined input that can change a force curve measurement parameterduring data acquisition.

In yet another aspect of the invention, a method of making a force curvemeasurement on a sample includes the step of providing an probemicroscope having a probe. Next, the method produces relative motionbetween the probe and the sample in response to a user-defined inputdefining intended motion in a direction substantially orthogonal to asurface of the sample. In addition, the method includes detecting therelative motion and comparing the relative motion to the correspondingintended motion. Further, motion of the probe is measured when thesample interacts with the probe, and a measurement parameter(s) can bechanged in response thereto.

According to another aspect of the invention, a method of making a forcecurve measurement on a sample includes the steps of generating a drivesignal to modulate a separation between the probe and the sampleaccording to a user-defined input. In addition, the method includesmeasuring the separation and controlling the drive signal in response tothe measuring step. Thereafter, motion of the probe is detected inresponse to the generating step. The method then includes changing ameasurement parameter in response to the detecting step.

These and other objects, features, and advantages of the invention willbecome apparent to those skilled in the art from the following detaileddescription and the accompanying drawings. It should be understood,however, that the detailed description and specific examples, whileindicating preferred embodiments of the present invention, are given byway of illustration and not of limitation. Many changes andmodifications may be made within the scope of the present inventionwithout departing from the spirit thereof, and the invention includesall such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

A preferred exemplary embodiment of the invention is illustrated in theaccompanying drawings in which like reference numerals represent likeparts throughout, and in which:

FIGS. 1A-1E illustrate a probe of an SPM as it is actuated to approachand retract from a surface so that the tip-sample forces can be mapped;

FIG. 2 is a plot illustrating force data obtained by the operation shownin FIGS. 1A-1E;

FIG. 3 is a partially broken away front elevation view of a DNA samplebonded between an AFM tip and a substrate;

FIG. 4 is a front elevation view of a standard SPM including aconventional piezoelectric tube actuator;

FIG. 5A is a plot illustrating a cyclical drive signal having aparticular amplitude and speed for making a force curve measurement;

FIG. 5B is a plot similar to FIG. 5A illustrating a cyclical drivesignal, characterized as including a pause between changes in thedirection of actuation of the Z piezo;

FIG. 6 is a diagram illustrating a force scanning probe microscope(FSPM) system according to a preferred embodiment of the invention,including a sensored scanner, a control system that maximizes forcecurve measurement flexibility and a mechanical feedback user interface;

FIG. 7 is a diagram further illustrating the FSPM of the preferredembodiment, including a force mode controller feedback system;

FIG. 8 is a side elevation view of the FSPM of the preferred embodiment,illustrating the Z sensor;

FIG. 9 is a cross-sectional front elevation view of the forcespectroscopy scanner shown schematically in FIGS. 6, 7 and 8;

FIG. 10 is a cross-sectional schematic view of a typical sensored Zstage preferably implemented in the scanner of FIG. 9;

FIGS. 11A-11C illustrate alternate embodiments of the configuration ofthe scanner of the FSPM of FIGS. 6-10;

FIG. 12 is a flow diagram illustrating a method of automaticallyeffecting a force curve measurement with a selected position gradient;

FIG. 13A is a plot illustrating a user-defined tip-sample separationgradient used to drive the Z piezo;

FIG. 13B is a plot illustrating a force versus time curve associatedwith actuating the Z piezo as shown in FIG. 13A;

FIG. 13C is a force curve generated by combining the position andcantilevered deflection time dependent plots shown in FIGS. 13A and 13B,respectively;

FIG. 14 is a flow diagram illustrating a method of driving a force curvemeasurement with a selected force gradient;

FIG. 15A is a plot illustrating a user-defined force gradient used tocontrol the actuation of the Z piezo;

FIG. 15B is a plot illustrating a Z piezo position profile that resultsfrom the force gradient input shown in FIG. 15A;

FIG. 15C is a force curve generated by combining the time dependentcurves shown in FIGS. 15A and 15B;

FIG. 16 is a flow diagram illustrating a method of automatically drivinga force curve measurement according to one or more trigger conditions;

FIG. 17 is a plot illustrating trigger events that cause a change in thedrive signal during a force measurement operation, according to thepreferred embodiment;

FIG. 18A is a plot illustrating an example of a force gradient such asthat shown in FIG. 15A;

FIG. 18B is a plot illustrating the Z piezo movement that results byinputting the user-defined force gradient shown in FIG. 18A for a hardsurface; and

FIG. 18C is a plot illustrating the Z piezo movement that results byinputting the user-defined force gradient shown in FIG. 18A for a softsurface.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Turning initially to FIG. 6, a force scanning probe microscope (FSPM)100 that provides highly accurate force measurements, including forcevolume measurements, with a high degree of data acquisition flexibilityis illustrated. FSPM 100 includes an atomic force microscope (AFM) 102,a data acquisition and control system 104, and a user interface 106.Probe microscope 102 includes a probe 108 having a cantilever I 10extending from a substrate 112. Cantilever 110 includes a free end towhich a tip 114 is coupled so that it extends generally orthogonal tocantilever 110. Probe 108 is placed in a support (not shown), such as aconventional probe holder, thus collectively defining a probe assembly.

Next, a sample 116 is mounted on a piezoelectric scanner 118 in aconventional fashion. The sample 116 may, for instance, comprise asingle molecule of a substance of interest. In the preferredimplementation, scanner 118 is used to actuate the sample in threesubstantially orthogonal directions, X, Y, and Z. Notably, movement inthe X,Y directions defines a scan plane generally parallel to themeasured surface of the sample 116, and movement in Z is substantiallyorthogonal to the scan plan. An important aspect of making the forcemeasurements contemplated by the present invention is preciselymodulating the tip-sample separation so the two interact at a particularscan location (x, y). In this regard, some combination of moving boththe tip and the sample could be implemented for providing the relative3-D movement.

According to the preferred embodiment, and as described in furtherdetail below, scanner 118 includes an X-Y tube scanner 120 that ismounted to the AFM chassis (160 shown in FIG. 8) and moves sample 116 inthe scan plane, i.e., generally parallel to a top surface of scanner 118and the sample surface. As a result, tube 120 operates to position aselected point of the sample beneath, and in-line with, tip 114 of probe108. Scanner 118 also includes a sensored Z actuator 122, which issupported by X-Y tube 120, and shown schematically in FIG. 10.

During FSPM operation, sensored Z actuator 122 translates sample 116towards (“approach”) and away from (“retract”) tip 114 of probe 108causing interaction between sample 116 and probe 108. In that regard, asprobe tip 114 and sample 116 interact, cantilever 110 deflects. Thisdeflection can be measured as a function of Z movement provided byscanner 118 (for example, one or more approach/retract cycles), and thecorresponding data can be used to plot a force curve.

The preferred method by which the deflection is measured is by employinga deflection detection apparatus 123 that directs a light beam “L”generated by a source 124 towards the back of cantilever 110. The beam Lis then reflected off cantilever 110 and towards a detector 126. Thedetector 126 can be, for example, a conventional four-cell photodetectorthat generates a deflection detection signal based on the position ofthe reflected beam. This signal is then transmitted to data acquisitionand control system 104 that communicates with scanner 118 in a closedloop configuration to modulate the tip-sample separation based on aparticular set of imaging parameters, as described in further detailbelow. Preferably, the light beam is produced by a low noise, lowcoherence length light source 124 (i.e., laser or super luminescentdiode).

Notably, such a probe microscope may be operated in a mode where someother probe motion besides deflection (e.g., oscillatory motion) ismeasured, or wherein the motion (i.e., deflection) of the probe ismaintained at some setpoint by application of some external force (i.e.,laser pressure), and thus the magnitude of this external force isrelated to the tip-sample interaction.

As described in greater detail below in conjunction with FIGS. 9 and 10,X-Y scanner 120 is preferably a piezoelectric tube scanner that iscoupled to sensored Z actuator or stage 122 via an appropriate coupling.Sensored Z actuator 122, on the other hand, is preferably apiezoelectric flexured stage actuator constructed from a metal mass andincluding flexure points that provide constrained motion in the intendeddirection, i.e., in the vertical or “Z” direction in this case. Thecombination of a tube scanner and a flexure stage actuator providessignificant advantages in making force measurements, including scanrange, predictable, repeatable motion, and high resonance frequency,detailed below.

With continued reference to FIG. 6, data acquisition and control system104 communicates with scanner 118 to actuate the scanner according toparticular force measurement parameters. More particularly, controlsystem includes a force controller 128, an AFM controller 130 and acomputer 132. Force controller 128 operates to modulate tip-sampleseparation and ensure that the desired Z actuator motion is beingmaintained via feedback from a sensor (162 in FIG. 8) that monitorseither the actual motion of sample 116, or the tip-sample separationdirectly. Force controller 128 is also coupled to SPM controller 130that controls motion of scanner 118 to image surface characteristics,for example, to obtain a topography image using a selected SPM mode ofoperation (e.g., oscillating mode).

Force controller 128 and SPM controller 130 further communicate withcomputer 132 which provides, in at least some embodiments of the presentinvention, instructions to controllers 128, 130 according to desiredexperiments. In general, desired force measurement parameters arecommunicated to data acquisition and control system 104, so as toachieve flexibility in making force measurements on a wide range ofsamples (see FIGS. 13A-C, 15A-C, and 17 and their correspondingdescriptions) and acquire a permanent record of the data.

Note that the exact nature and routing of the signals and the variouscontrollers to which are referred are relatively unimportant. Forinstance, control and data channels could be transmitted as analog ordigital signals. The separation of control functions into three nominalcontrollers/computers could just as well be accomplished with a singlecontroller/computer in hardware or software. In short, those skilled inthe art could readily conceive other schemes of implementation for thecontrol and data acquisition algorithms described which would still notdeviate from the spirit and scope of the underlying inventive concept.

In this regard, force measurement parameters may be modified accordingto communications from user feedback interface 106 to either computer132 for automatic control, or directly to force controller 128 formanual control. For example, the control provided by control system 104may be defined by a user waveform input 134, or a user trigger input136, according to the operator's desired force measurement, as describedin detail below in conjunction with FIGS. 12-17. On the other hand,rather than providing computer control of desired force measurementparameters, these parameters can be communicated directly with the fourcontroller 128 via a mechanical feedback user interface 140.

Mechanical feedback user interface 140 of system 100 is coupled to forcecontroller 128 to allow the user to manually adjust the actuation ofscanner 118. Therefore, the user can correspondingly adjust thetip-sample separation. Mechanical feedback interface 140 preferablyincludes a manipulatable device such as a rotary knob 142 which a usermanually manipulates to displace the sample (or probe as appropriate forthe particular implementation) to cause sample 116 to interact with tip114 of the probe 108. In this way, the user can essentially “feel” thesample structure and properties associated with sample 116, based on ameasured force that is fed back to manual interface 140 as an alert. Inthe preferred embodiment, this alert signal can be used to alter acharacteristic associated with manipulating knob 142, e.g., torque. Theoperator can then adjust tip-sample separation to stretch molecules,observe unfolding/refolding of protein domains, etc. Manual interface140 preferably also includes a display 144 so the FSPM operator canmonitor quantitative values associated with the forces being sensed.

Still referring to FIG. 6, computer 132 is coupled to a display 138 topresent the actual force, force volume, or SPM image data to the FSPMoperator. Moreover, computer 132 of system 104 communicates with akeyboard/mouse or other suitable user interface 146 to allow thesevarious options to be selected by an operator via, for example, agraphical user interface (GUI) (not shown).

Turning to FIG. 7, FSPM 100 is shown in further detail. In particular,the force feedback mechanism of the preferred embodiment is illustrated.Force controller 128 of data acquisition and control system 104 includesa Z position drive/feedback loop 148 including a closed-loop controlblock 150, preferably, conventional analog circuitry including acomparator and a gain stage (not shown), that generates a drive signalthat controls piezoelectric flexured Z actuator 122 to maintain linearmotion of Z actuator 122; in other words, motion corresponding to thedesired Z motion as defined by the user input. Control block 150generates Z-stage drive signal based on two inputs, a first being theactual Z motion measured by sensored Z actuator 122, and the secondbeing the desired Z actuator motion transmitted automatically fromcomputer 132 as a Z position input waveform 154 as detailed below, ormanually from manual input device 140.

With respect to the desired Z actuator motion, flexibility in makingcorresponding force curve measurements is achieved by controlling thetip-sample separation via Z actuator 122. This motion of Z actuator 122can be defined by a standard input for making force curve measurements,such as a cyclic triangle wave, as described previously (FIG. 5A), or itcould be a more complex user-defined input. For example, a deflectionforce feedback block 152 may be employed in conjunction with the Zposition waveform 154 to maintain a particular force or force profile,or to change a force measurement parameter in response to a “trigger”condition, as shown and described in conjunction with FIGS. 14, 15A-C,16 and 17. Alternatively, the Z position waveform block 154 can input anappropriate profile to computer 132 that can then be communicated toforce controller 128 to define a predetermined Z position profile overtime (FIGS. 12 and 13A-C). In yet another alternative, rather thanautomatically controlling Z actuator 122 to make selected forcemeasurements, the desired actuator motion may be controlled manuallyduring acquisition of force data by an operator via manual input device140.

Referring to FIGS. 6 and 7, in operation, as tip-sample separation ismodulated (in the preferred embodiment, by actuating Z actuator 122),probe 108 interacts with sample 116. As a result, the motion ofcantilever 110 of probe 108 changes and this change in motion isdetected by detector 126. Detector 126, as described previously,generates a probe motion signal (for example, if detector 126 is a splitphotodiode, the quantity (A−B)/(A+B) defines the deflection) that istransmitted to computer 132. Computer 132 includes a detectormeasurement circuit 158 that receives the deflection detection signaland, in response, determines the force acting on probe 108. Deflectionforce feedback block 152 of computer 132 then determines whether aparticular force is being maintained (force profile input), or whether atrigger condition is satisfied. In response, feedback block 152generates and transmits an appropriate control signal to forcecontroller 128 (FIG. 6), which generates a Z-stage drive signal tocontrol actuator 122 translation according to the user's specificationsfor that particular force measurement, i.e., to maintain or change theforce measurement parameters, such as direction of Z motion, speed,etc., as described further below. Computer 132 may also include one ormore Z-position input waveforms that may be selected by an operator(again, preferably via a GUI) to allow the user to flexibly control theactuation of the Z piezo according to a particular experiment to beperformed (FIGS. 12 and 13A-C).

The position input to control block 150 input is the actual motion of Zactuator 122, as measured by a sensor (162 in FIG. 8). During a forcemeasurement, the sensor measures the motion of Z actuator 122 andgenerates an associated Z actuator position signal that it transmits tocontrol block 150 of force controller 128. Control block 150 thendetermines whether the actual translation of Z actuator 122 is the sameas (i.e., linear with) the instructed Z actuated movement provided byeither computer 132 or manual input device 140. If not, control block150 generates an appropriate Z-stage drive signal to correct any Ztranslation that does not correspond to the user's input. In this way,precision in modulating the tip-sample separation is maintained.

In FIG. 8, the sensor feature aspect of scanner 118, and in particular Zactuator 122, is shown. SPM 102 of system 100 includes a chassis 160 towhich the deflection detection system 123 and the probe assembly,including probe 108 and mount 117, are coupled. Chassis 160 alsosupports scanner 118, which is fixed thereto. Again, the X-Y translationof scanner 118 is provided by piezoelectric tube scanner 120, while theZ translation is provided by flexured piezoelectric actuator 122. Tomonitor the translation of Z actuator 122, a sensor 162 is preferablycoupled to Z actuator 122. Ideally, sensor 162 is a capacitive sensorthat measures the translation of the Z actuator 122 (and, hence,translation of the sample 116) by measuring the capacitance changecaused by a change in the separation of the plates of the capacitor.Referring briefly to FIG. 10, one arrangement of the plates ofcapacitive displacement sensor 162 is shown. An upper plate 163 is fixedto a stationary portion of flexure stage 122, while a lower plate 164 isfixed to a translatable portion 168 of stage 122. As a result, when theZ actuator 122 is actuated, movement of translatable portion 168 (andthus sample 116) causes the perpendicular separation between plates 163,164 to be modified. This change in separation is measured to determineactual Z translation causes a corresponding change in the measuredcapacitance. Notably, because the flexure stage from the Z actuator 122is robust, the separation between plates 163, 164 along their surfaceareas remains constant (i.e., they maintain their parallel relationship)when Z actuator 122 is actuated. Moreover, although a parallel platecapacitor is preferred, sensor 162 could be an alternative type ofsensor such as a piezo-resistive sensor mechanically coupled to areference and the translatable portion of the piezoelectric Z-stage(i.e.—LVDT, or strain gauge sensors).

As noted above, inaccuracies in tip/sample positioning can arise due toresidual mechanical effects such as hysteresis, creep, thermalexpansion, etc. that can act on all or part of the AFM system. Because,in making force measurements, it is the tip-sample separation that iscritical, an arrangement that eliminates or minimizes these effects wasdesired. In this regard, an alternative to the sensor arrangementdescribed above, also shown in FIG. 8, includes coupling a total systemproximity sensor 165, such as a capacitive sensor, between the sampleand the probe itself to provide a direct measure of tip-sampleseparation. As a result, by measuring the actual tip-sample separationbetween sample 116 and tip 114 with sensor 165, position feedback loop148 (FIG. 7) maintains a high degree of positioning accuracy. Such atotal system proximity sensor 165 essentially eliminates the effects ofhysteresis, creep, and thermal expansion because these effects occurwith respect to the sample and probe assembly simultaneously. If theparallel plates of a capacitive sensor are coupled to the sample andprobe assembly, respectively, these potentially damaging effects canceleach other out when tip-sample separation is measured.

Next, a detailed view of force spectroscopy scanner 118 is shown in FIG.9. Scanner 118 provides movement of a sample (116 in FIG. 6, forexample) in three orthogonal directions, which we refer to hereinafteras movement in X, Y and Z. Note that scanner 118 operates to move sample116 underneath probe 108 (FIG. 6), and that movement in X and Y definesa plane generally parallel to a surface of the sample. Again, althoughforce measurements are made at a particular sample location (x,y),movement in the XY plane is required for analyzing different regions ofthe sample. By generating force curves at a variety of XY locations ofthe sample, a force volume image may then be generated, for example.

Scanner 118 includes a scanner mounting base 169 that is coupled to thechassis (160 in FIG. 8), and that serves as a reference for positionmeasurements. A scanner core 172 extends upwardly from scanner mountingbase 169 and is coupled thereto. Scanner core 172 defines a tubularstructure that encloses and protects piezoelectric XY tube scanner 120,as well as flexured Z actuator 122. Scanner core 172 is preferably madeof a metal, ideally a commercially available steel such as INVAR.

Z actuator 122 is coupled to XY tube scanner 120 via an XY-Z coupling174. XY-Z coupling 174 is a cap that is positioned over the top of tubescanner 120 and that provides a mounting surface 176 for fixing flexuredZ actuator 122 to XY tube scanner 120 conventionally. When the actuator122 is assembled, axes passing through the center of each of the coupledscanner portions are generally collinear.

In this arrangement, translation of XY tube scanner 120 causescorresponding movement of flexured Z actuator 122. Notably, bypositioning tube scanner 120 beneath Z actuator 122 the X-Y movementproduced by tube scanner 120 is amplified at a free end 178 of scanner118.

Notably, a conventional piezoelectric tube scanner, such as scanner 120employed in the preferred embodiment, provides a limited range ofmotion, such that the scan range (i.e., in the XY plane) provided byscanner 118 alone is less than ideal. However, in the preferred designshown in FIG. 6 (and in more detail in FIG. 9), greater XY scan range isachieved due to the fact that the object being moved by scanner 118 isdisplaced from tube scanner 120 a distance generally equal to the lengthof sensored Z actuator 122, which is supported by tube scanner 120. Inother words, scanner 118 mechanically amplifies the XY range afforded byscanner 118.

It follows that the larger distance between tube 120 and the objectbeing moved, in this case a sample placed at the free-end 178 of scanner118, the larger the amplification. In this regard, to further amplifymovement of free end 178 of the scanner 118 produced by the XY tubescanner, a cylindrical Z extension 180 can be included, as shown in FIG.9. Z extension 180 has opposed ends including a first end 182 which isfixed to a top portion 186 of Z actuator 122 via a mounting flange orring 188, and a second end 184 that is configured to support a sampleholder 185. As with the X-Y tube scanner 120 and Z actuator stage 122, Zextension has a central axis collinear with the central axis of scanner118. The sample holder is conventional in the art, such as that shown inU.S. Pat. No. Re 34,485. As with piezoelectric Z actuator 122, Zextension 180 is preferably made of INVAR. To minimize the mass ofextension 180, and thus prevent compromising the strength of scanner118, a plurality of holes 190 are formed therein.

In FIG. 10, a typical flexured Z actuator stage 122 of forcespectroscopy scanner 118, which is adapted for providing Z positionfeedback as described previously, is shown schematically. Z actuator 122includes a mounting surface 192 that is coupled to X-Y tube scanner 120as shown in FIG. 9 with XY-Z coupling 174. Z actuator 122 is preferablya metal block (e.g., Invar) having portions thereof removed to create afixed section or frame 166 and a translatable section 168 coupled toprovide constrained motion in Z, i.e., in a direction perpendicular tosurface 120. Linking fixed and translatable portions 166, 168,respectively, are weakened points that permit movement of the metal masswhen forces are applied thereto. In particular, the metal block includesa series of flexure points 194, 196, 198, 200 formed to constrain motionof translatable portion 168 in a plane orthogonal to the vertical or Zdirection (i.e., in the XY plane), while allowing motion of portion 168in the vertical direction.

More particularly, each of the flexure points 194, 196, 198, 200comprises a web of metal that can “flex” to allow a sample (116 in FIG.6) mounted on a translating/mounting surface 170 of translatable portion168 to be translated in a direction orthogonal to mounting surface 170.Again, flexure points 194, 196, 198, 200 are coupled to fixed portion166 and support center translatable section 168 of scanner 118.

Translatable section 168 defines mounting surface 170 and also defines alower contact surface 202 that interfaces with, preferably, a piezostack 204 mounted within Z actuator intermediate a surface 206 of fixedportion 166 and contact surface 202. Piezo stack 204 is a conventionalpiezoelectric component that produces motion in a selected direction inresponse to appropriate voltages applied to electrodes placed on thepiezoelectric material of piezo stack 204. Piezo stack 204 expands andcontracts in response to the applied voltage signals such that themechanical motion is transferred to center section 168 of Z actuator 122via surface 202. Section 168, in turn, moves vertically as flexurepoints 194, 196, 198, 200 flex. In this case, piezo stack 204 isconfigured to move in a direction substantially orthogonal to mountingsurface 120 in response to the control voltages.

As noted above, to measure the motion in Z provided by actuator 122,first plate 164 of capacitive displacement sensor is fixed totranslatable section 168 of Z actuator 122, while opposed plate 163 ismounted to a surface 165 of fixed portion 166 of Z actuator 122. As aresult, movement of translatable section 168 relative to fixed portion166 can be precisely measured as a change in capacitance due to a changein the perpendicular separation “D” of plates 163, 164 of capacitivedisplacement sensor 161. More particularly, capacitance is proportionalto one over the separation distance, i.e., C=εA/D, where “D” is theperpendicular distance between the parallel plates, thus providing ameasure of Z translation. Notably, plates 162, 164 of sensor 161preferably are rings.

In operation, a displacement signal is generated by sensor 161 and fedback to Z position feedback loop 148 (FIG. 7) to determine whether thevoltage applied to piezo stack 204 resulted in the intended motion. Ifnot, one or more a correction Z-stage drive signals can be generated toprovide the intended motion of sample 116. Again, this aspect of thepreferred embodiment allows precise control of the scanner which iscritical to achieving flexibility in making force measurements, to bedescribed below.

Notably, flexured Z actuator 122 shown in FIG. 10, is configuredaccording to the schematic shown in FIG. 8. However, as noted above,when a sample coupled to surface 170 is translated in the verticaldirection, effects due to non-linearity, hysteresis, creep, drift, etc.,can contribute to the Z motion provided by actuator 122, thus causingpositioning/spacing problems. Therefore, a sensor arrangement as shownin phantom in FIG. 8 may be preferred for measuring actual Z motionunder certain environmental conditions, conducting particularexperiments, etc.

Scanner 118 also provides a high resonant frequency, three-dimensionalactuator that achieves optimum performance in x,y scan range and true Zmotion in a small package. In the latter regard, the height of sensoredZ actuator 122 is approximately 2″ such that once the actuator 122 iscoupled with piezoelectric XY tube scanner 120, scanner 118 ismaintained in approximately the same package as a conventional XY-Z tubescanner.

Overall, scanner 118 produces large Z position range, with substantialX-Y range, while being contained in a small package. Moreover, scanner118 is readily adapted for Z sensing. As a result, scanner 118 isadapted to provide closed loop monitoring of tip-sample separation,while minimizing noise problems and having a high resonant frequencywith respect to Z positioning (mechanical flexure driven by piezo versusa simple piezo stack or tube), which is particularly important whenmaking force measurements.

Next, alternative configurations of force spectroscopy scanner 118 areshown in FIGS. 11A-11C. In FIG. 11A, a scanner 210 includes a sectionedZ actuator (tube or stack) 212 that is disposed on top of a flexured Zactuator 214 which, in turn, is mounted on an XY tube 216. Because XYtube 216 is arranged at the bottom of scanner 210, a relatively widerange of XY scanning capability is achieved, for reasons describedpreviously. Z tube 212 is adapted to accommodate a sample (not shown)and provides motion generally orthogonal to a surface of the sample tomodulate tip-sample separation according to the user's requirements. Inthis arrangement, flexured Z actuator 214 may provide coarse adjustmentof tip-sample separation, while Z tube 212 can be implemented to providea fine adjust of the tip-sample separation, i.e., movement of thesample. This may be particularly desirable when working with delicatesamples. Or, Z tube could be driven by a high frequency oscillation toaccommodate different modes of operation such as a mode similar to thatdescribed in the literature as “fly-fishing” in which the tip isoscillated at a relatively small amplitude and relatively high frequency(via acoustic or magnetic “AC” or TappingMode) while the tip-sampleseparation is reduced, with the desired result being that the probe“snags” or “catches” a single molecule on the surface. The addition of asecond Z axis piezo would allow a similar technique where the user caninstead use the second piezo to provide the low amplitude/high frequencyoscillation.

In the alternative shown in FIG. 11B, scanner 220 includes a flexured Zactuator 224 that is positioned intermediate a sectioned piezoelectric Ztube 226 and a piezoelectric XY tube 222 on which the sample resides.Unlike the previous cases, in this arrangement, the Z actuators 224, 226move the XY tube actuator 222. Therefore, scanner 220 provides theflexibility of the scanner shown in FIG. 11A. However, XY range ofscanner is compromised, as it is disposed nearest the object to betranslated, i.e., the sample. Finally, as shown in FIG. 11 C, a similararrangement of a flexured Z actuator 236, an XY tube actuator 234, and asectioned Z tube actuator 232 is shown. In this case, flexured Zactuator 236 is fixed to the SPM chassis to provide an actuator havingsuperior strength to the previous embodiments. Moreover, XY scanningrange is larger than scanning range of actuator 220 in FIG. 11B due tothe fact that the perpendicular distance between the XY tube scanner 234and the sample (disposed on the sectioned Z tube actuator) is larger. Ineach of the alternative configurations in FIGS. 11A-11C, to bestdetermine tip-sample separation, the preferred Z sensing method includesutilizing sensor 165 shown in phantom in FIG. 8, where a direct measureof separation is made.

By utilizing the sensors of the preferred embodiment, FSPM 100 preciselycontrols the Z movement of the actuator 118, monitoring whether the Zmovement is in the intended direction, such that significant flexibilityin making force curve measurements can be achieved. In particular,according to a further aspect of the preferred embodiment as shown inFIGS. 12-17, methods are disclosed for altering a force measurementparameter based on a selected user-defined input.

Turning initially to FIG. 12, a method 250 of making a force curvemeasurement by driving the tip-sample modulation with a tip-sampleseparation gradient includes, initially, a start-up and initializationBlock 252. Next, in Block 254, a signal corresponding to a first pointof a defined input (in this case, a tip-sample separation gradient) istransmitted to the force controller (128 in FIG. 7). Then, method 250generates a drive signal based on the tip-sample separation gradient forthat point in Block 256. This drive signal is then applied, in Block258, to the scanner (118 in FIG. 6).

Method 250 then measures the Z position of the sample in Block 260 (FIG.8). Next, in Block 262, the method determines whether the movement in Zcorresponds to the user-defined input (i.e., closed-loop Z-positioning).If not, a new drive signal to correct the Z motion is generated in Block264 and method 250 is returned to Block 258 to apply the new drivesignal to the scanner. If, on the other hand, movement corresponds tothe user-defined input, cantilever deflection is measured in Block 266.This data is collected and stored in Block 268 and then plotted as aforce versus time curve for that point. Method 250 then returnsoperation to Block 254 to transmit a signal corresponding to theuser-defined input for the next point of the position gradient. Bycombining the position gradient and the force gradient (Block 258) aforce versus separation profile, i.e., a force curve is generated inBlock 270.

An example of method 250 in operation is shown in FIGS. 13A-13C. Thewaveform shown in FIG. 13A corresponds to user-defined input of Block254. And, the corresponding forces between the AFM probe tip and thesample generated as a result of this position profile are measured andplotted in FIG. 13B (Block 268). Notably, the velocity of this actuationis defined by the slope of the separation curve. Moreover, note that thezero (“0”) piezo position corresponds to zero tip-sample separation, andthat negative slope indicates movements upwardly, i.e., towards theprobe as an increasing negative Z-position. A piezo position at or below“0” indicates no separation as sample engages the AFM tip and continuesto move, potentially causing the cantilever to deflect while in contactwith the sample. In that case, the tip of the probe may or may not bepenetrating the sample, as may be indicated by the measured forces (FIG.13B). On the other hand, as the Z-piezo moves the sample downwardly(positive slope in FIG. 13A), tip-sample separation is increasing.However, as the Z-piezo is withdrawn past the zero position (initialtip-sample contact), the sample may bind to the tip such that there iszero actual tip-sample separation for a time as the cantilever deflectsto follow the downward motion of the sample.

Referring collectively to FIGS. 13A and 13B, as the sample approachesthe tip of the cantilever starting at time t₁, the tip experiences aforce (FIG. 13B) at about t₂ where the cantilever begins to deflectupwardly, which is correspondingly sensed by the deflection detectionsystem (see 123 in FIG. 6). The flexured Z actuator 122 (FIGS. 6-11) isthen caused to translate further in the same direction, i.e., towardsthe fixed probe, and at the same velocity (constant slope). Thismovement of the flexured Z actuator is halted at a time t₃ where theprobe experiences a positive deflection or force F₁ as shown in FIG.13B. After holding the Z position constant for a period of time t₄ minust₃ (for example, to allow binding of a molecule to the tip), thedirection of movement of the flexured Z actuator is reversed as thesample is pulled away from the tip. In this case, the positivedeflection of the cantilever is reduced as it passes through the zeropiezo position (time t₅) where the tip is essentially resting on thesample as the Z actuator pulls the sample further away from the probe.As this actuation continues, binding between the tip of the probe andthe sample produces a negative deflection and a corresponding negativeforce that increases to a value F₂ (FIG. 13B), as Z translationcontinues until a time t₆. Notably, if there was no binding of thesample to the tip, the actual separation between the sample and the tipwould be non-zero.

At time t₆, this negative deflection of the probe decreases as thedirection of translation of Z motion is reversed, such that thedeflection of the cantilever of the probe again approaches zero. In thiscase, in the case of a titin molecule, the molecule is allowed to“refold,” as Z actuator again moves in a direction towards themicroscope tip. The corresponding measured forces are indicative ofsample properties. This approach/withdraw cycle is repeated until thepiezo is translated away from the probe at a constant velocity at timet₁₁. As a result, the Z actuator moves the piezo until the tip releasesfrom the sample, at which time t₁₂ the force plotted in FIG. 13B returnsto zero, i.e., as the cantilever of the probe returns to its free-airdeflection.

Thereafter, the position gradient shown in FIG. 13A and thecorresponding force data measured as a function of time (FIG. 13B),which again is measured in response to the selected tip-sampleseparation gradient shown in FIG. 13A, are combined in conventionalfashion to produce the force versus separation curve shown in FIG. 13C.In sum, by controlling the Z actuator position relative to the fixedprobe, different properties of the sample supported by the flexuredactuator can be observed and recorded according to the user'srequirements. For example, in one experiment, forces measured during thestretching and refolding of particular molecules, such as titinmolecules, can be analyzed according to particular models of theirmechanical behavior.

FIG. 13C illustrates a force curve similar to that shown in FIG. 2. Notethat the bi-directional arrows on the force curve indicate approach(decreasing “piezo Z”, i.e., decreasing tip-sample separation) andretract (increasing “piezo Z”). As the sample approaches the cantilevertip, zero force is experienced by the tip such that no deflection of thecantilever is detected. As the tip begins to interact with the sample atposition z₁ (generally corresponding to time t₂ in FIGS. 13A and 13B)the cantilever begins to deflect upwardly. This deflection is plotted asa positive force. As the sample is translated further towards the tip,the cantilever of the probe deflects further, thus increasing the forcedetected. As the tip continues to interact with the sample, the actuatorposition reaches a point Z₂ where the direction of the movement ischanged. In particular, the sample is pulled away from the tip, thuscausing the measured force to decrease until it reaches a point z₁ wherethe probe experiences zero force once again.

As the sample is withdrawn further from this zero force position,because in this case the tip binds to the sample, the sample begins to“pull” the probe downwardly as the force continues to increase in theopposite direction. Thereafter, the tip releases from the sample at Z₃,such that the probe deflection returns to its free-air zero value.

Another user-defined profile is illustrated in FIG. 14 as a method 300for controlling one or more force measurement parameters according to auser-defined force gradient. The method 300 is also illustratedgraphically with an example in FIGS. 15A, 15B and 15C. After a start-upand initialization Block 302, method 300 transmits a user-defined forcegradient signal for a particular point of the gradient to the forcecontroller (128 in FIG. 7) in Block 304. Next, in Block 306, the forcecontroller generates a drive signal based on the user-defined forcegradient for that point (e.g., selecting a velocity, direction andduration of Z actuation), and then applies the drive signal to the Zactuator in Block 308. As the drive signal is applied, the Z position ofthe scanner is measured in Block 310 (closed-loop Z-positioning), andmethod 300 determines whether the movement corresponds to that dictatedby the drive signal in Block 312. If not, a drive signal is generated inBlock 314 to correct the Z motion. If so, the deflection of thecantilever is measured, collected and stored to determine the force onthe cantilever in Block 316.

Method 300 next plots position on a position versus time plot for thatpoint in Block 318. In Block 320, method 300 determines whether theforce on the cantilever corresponds to the user-defined force input(FIG. 15A, for example, described below) for the particular pointdefined in Block 304. If the force correspondence requirement is notmet, the process is returned to Block 306 to generate a new Z-stagedrive signal based on the user-defined input and the measured deflection(instruction generated and communicated from deflection force feedbackblock 150 in FIG. 7). For example, if the measured force is less thanthe desired force, a signal is transmitted to the force controller toinstruct the force controller to transmit an appropriate signal to the Zactuator to move the sample faster so that the correct cantileverdeflection (i.e., force) for that point is achieved. If, on the otherhand, the force is met, method 300 asks whether each point in the forcegradient has been considered in Block 322. If not, the process returnsto Block 304 to transmit a force control signal for another point of theforce gradient. If data has been obtained for each point in the forcegradient, the collected and stored data (including the force gradientand position plot) are combined and plotted as a force versus a positionprofile of the sample (i.e., the force curve) in Block 324.

An illustrative example of method 300 in operation is shown in FIGS.15A-15C, defining another type of user-defined waveform input to controlthe acquisition of a force curve. Again, method 300 is directed toinputting a force gradient (FIG. 15A) (i.e., a rate of change of force)and measuring the corresponding position of the piezo required toachieve that force gradient (FIG. 15B). More particularly, withreference to FIG. 15A, between time t₀ and time t₁, the force is heldconstant at a zero value which typically will correspond to bringing thetip and sample into contact, as shown in FIG. 15B for this time range.At time t₁ the force gradient in FIG. 15A instructs the Z piezo actuatorto move so that the force increases linearly from time t₁ to time t₂.Notably, as shown in FIG. 15B, at t₁ the cantilever deflection is zeroand the piezo position is at zero tip-sample separation. Then, thecantilever deflects upwardly during the time t₂ minus t₁ as the actuatormoves further towards the probe with the tip in contact with the sample.Between t₂ and t₃, the force is held constant, and thus the forcecontroller does not cause the Z actuator to move.

At t₃, the method 300 instructs the Z actuator to move the sample sothat the force is reduced linearly. At the time t₄, the force is zerowhile force controller continues to cause the Z actuator to pull thesample away from the microscope tip. Then, between t₄ and t₅, a linearforce gradient in the same direction provides the instructions to theforce controller for actuating the flexured piezoelectric Z actuator. Itis notable that the actuation of the Z actuator required to maintain thelinear force gradient shown between t₄ and t₅ in FIG. 15A, is nonlinearas shown in FIG. 15B. In other words, force is not directly proportionalto tip-sample separation. As a result, including such a force gradientcan provide useful information that cannot be obtained by using thetip-sample separation gradient shown in FIGS. 13A-13C. Finally, as thelinear force gradient is continuously applied, the Z-stage drive to theZ piezoelectric actuator continues to pull the sample away from the tipuntil point t₅ at which time the tip releases from the sample surfaceand the force on the probe returns to zero. As in FIGS. 13A-13C, thetime dependent curve shown in FIGS. 15A and 15B can be combined toproduce the force versus separation curve shown in FIG. 15C. A notableregion of the force curve of FIG. 15C is between the times t₄ and t₅(labeled (t₄, t₅)) illustrating the binding of the tip to the sample asthe Z-piezo is moved away from the probe. Although the force versusseparation curves shown in FIGS. 13C and 15C are similar, theinformation provided in the measured curves in FIGS. 13B and 15B caneach provide unique, valuable information regarding the particularexperiment being conducted.

Next, trigger operation of the force SPM is very similar to theuser-defined force gradient shown in FIGS. 13A-13C. However, rather thanbeing predetermined as is the case with the user-defined force gradient,trigger-based operation can alter the force gradient, real-time, inresponse to a particular condition.

Referring initially to FIG. 16, a method 400 of trigger operationincludes a start-up and initialization Block 402. Next, the Z-stagedrive signal is generated according to at least one force measurementparameter for a particular point (e.g., a particular point in time onthe trigger profile such as that shown in FIG. 17), in Block 404. Next,the Z-stage drive signal is applied to Z actuator to move the actuatorfor a particular amount of time, and in a selected direction and speedin Block 406. Next, in Block 408, method 400 measures the Z position ofthe sample to determine tip-sample separation. As part of theclosed-loop positioning, in Block 410, method 400 determines whether theZ position corresponds to the drive signal. If it does not, method 400generates a new Z-stage drive signal to correct Z motion so itcorresponds to the intended motion defined by Block 404. If, on theother hand, Z position corresponds to the drive signal, the cantileverdeflection for that point is measured and stored in Block 414. Next, inBlock 416, method 400 collects and stores the corresponding positiondata.

Continuing, method 400 next determines whether a trigger condition hasbeen met. If not, the process returns to Block 404 to generate anotherZ-stage drive signal for another particular point, i.e., the next pointin time. If, on the other hand, the trigger condition is met, method 400changes at least one force measurement parameter of the force curveacquisition process in Block 420. Once all trigger conditions of thetrigger profile (for example, FIG. 17) have been met, method 400 isterminated and a position versus time plot is combined with the triggerprofile to generate a force curve.

Turning to FIG. 17, a force versus time curve generated according to themethod 400 illustrated in FIG. 16 is shown. More particularly, as thecomputer instructs force controller to generate a Z-stage drive signal,the sample approaches the tip of the probe of the AFM and zero force onthe probe is measured by the deflection detection system. Thereafter, asthe tip begins to interact with the sample (e.g., a molecule) at timet₁, the force increases linearly to a value F₁ (positive deflectionforce) as the force spectroscopy actuator narrows the tip-sampleseparation.

At time t₂, a first trigger condition (a predetermined force) is met ata positive deflection force equal to F₁. In response, tip-sampleseparation is kept at the t₂ value for a time period t₃ minus t₂ (forexample, to allow time for the tip to bind to the sample). At t₃, basedon the first trigger, the computer transmits a signal to the forcespectroscopy scanner to begin withdrawing the sample from the tip toincrease tip-sample separation. At t₄, the tip of probe of FSPM isresting on the sample surface such that there is no deflection of thecantilever of the probe measured. As the separation is increasedfurther, a linear force gradient is measured during a time t₅ minus t₃as the tip passes through the zero force point at t₄. At that point,upon detection of the negative deflection force at t₅, Z movement of theforce spectroscopy scanner is halted for a period of time equal to t₆minus t₅ At t₆, computer instructs force controller to continue to“pull” on the sample based on the second trigger at a particularvelocity to produce the force gradient shown in region “A”. Tip-samplebinding deflects the cantilever downwardly, and these forces aremeasured between times t₆ and t₇, producing as described previouslyuntil the tip separates from the sample at t₇ and the force between thetip and sample is again returned to zero as the tip is no longerinteracting with the sample.

A straightforward example of using a force gradient to determine asample property is illustrated in FIGS. 18A-18C. FIG. 18A defines aforce gradient that is used to control the Z actuator similar to thatshown in FIG. 15A. For a hard surface, illustrated with the Z positionprofile in FIG. 15B, the Z actuator is caused to move in a generallylinear fashion both on the approach 450 (zero then increasing force) andretract 452 of the tip-sample separation. More particularly, to followthe force profile shown in FIG. 18A, the actuator moves linearly until atime t₁ defining a zero position where the tip begins to contact thesample, at which the time the force on the tip begins to increase. Inthe region from t₁ to t₂ the force gradient increases linearly (FIG.15A) and the actuation of the Z actuator moves linearly as well (FIG.15B) as the tip presses against the hard surface. At the peak force F₁(at time t₂) the actuation of the flexured Z actuator is at a peak Z₁such that the cantilever is deflected upwardly at its maximum. At thispoint, the instruction based on the force gradient is to actuate Ztranslation to reduce the force to a zero level. The motion of the Zactuator, in response, is linear to the zero point (time t₃) where thetip barely contacts the sample surface. As the sample is furtherwithdrawn from the tip, the force remains at zero as there is no bindingbetween the sample and tip.

In each of the methods illustrated in FIGS. 12, 14 and 16, it is notablethat the precise Z-positioning provided by scanner 118 of the preferredembodiment enables the use of a wide range of user-defined inputprofiles, thus allowing FSPM 100 to target measuring particularmechanical properties (e.g., based on sample models) of a wide range ofsamples.

In contrast, for a soft surface, as shown in FIG. 18C, to achieve thesame peak force F₁ on the tip of the probe, the force profile shown inFIG. 18A causes the Z piezo to move to a position Z₂ in a non-linearpath, whereby the value of Z₂ is much greater than Z₁, thus indicating asofter sample. As a result, with the same force profile, two types ofsamples can be investigated by considering their position profiles.

Although the best mode contemplated by the inventors of carrying out thepresent invention is disclosed above, practice of the present inventionis not limited thereto. It will be manifest that various additions,modifications and rearrangements of the features of the presentinvention may be made without deviating from the spirit and scope of theunderlying inventive concept.

1. A method of making a force curve measurement on a sample, the methodcomprising: providing a probe microscope having a probe; producingrelative motion between the probe and the sample in response to auser-defined input defining intended motion in a direction generallyorthogonal to a surface of the sample; detecting the relative motion andcomparing the relative motion to the corresponding intended motion;measuring a force on the probe when the sample interacts with the probe;and changing a force curve measurement parameter in response to saidmeasuring step.
 2. The method of claim 1, further comprising the step ofgenerating relative motion between the probe and the sample in responseto said detecting step.
 3. The method of claim 1, wherein said measuringstep includes detecting a motion of the probe.
 4. The method of claim 1,wherein said changing step is performed based on the user-defined input.5. The method of claim 1, wherein the force curve measurement parameteris a direction associated with said producing step.
 6. The method ofclaim 1, wherein the force measurement parameter is a speed associatedwith said producing step.
 7. The method of claim 1, wherein the forcecurve measurement parameter is pausing said producing step for apredetermined amount of time.
 8. The method of claim 4, wherein theuser-defined input is a path defined by force versus time.
 9. The methodof claim 8, further including the step of determining a force gradientbased on said measuring step performed for at least two points in time.10. The method of claim 1, wherein said producing step is interruptedprior to said changing step.
 11. The method of claim 1, wherein saidmeasuring step is performed for at least two points in time, and saidchanging step is performed in response to a predetermined change in theforce.
 12. The method of claim 1, wherein said changing step ispredetermined.
 13. The method of claim 12, wherein said changing stepincludes modulating a separation between the sample and the probe sothat the measured forces corresponding to a plurality of points in timecorrespond to a predetermined force profile as a function of time. 14.The method of claim 13, wherein said modulating step is performed usinga force feedback loop.
 15. The method of claim 1, further comprising thestep of measuring a separation between the sample and the probe as afunction of time.
 16. The method of claim 1, wherein said changing stepincludes controlling the separation between the probe and the sampleaccording to a user-defined path defined by position versus time. 17.The method of claim 1, wherein said producing step is performed by asensored Z stage.
 18. The method of claim 17, wherein said sensored Zstage is coupled to a piezoelectric tube scanner, said tube scannerproviding scanning motion in a plane substantially orthogonal to thethird axis.
 19. A method of making a force curve measurement on asample, the method comprising: generating a drive signal to modulate aseparation between a probe and the sample according to a user-definedinput; measuring the separation; controlling the drive signal inresponse to said measuring step; detecting a force on the probe inresponse to said generating step; and changing a force measurementparameter in response to said detecting step.
 20. The method of claim19, wherein the user-defined input is an automatic input.
 21. A methodof making a force curve measurement on a sample, the method comprising:measuring a force on a probe as the probe interacts with a sample inresponse to user defined input; comparing the force to a set-point;using a feedback loop to maintain the force at the set point; andadjusting the set-point according to the user-defined input.